Mitochondria are major suppliers of cellular energy in neurons; however, utilization of energy from glycolysis vs. mitochondrial oxidative phosphorylation (OxPhos) in the presynaptic compartment during neurotransmission is largely unknown. Using presynaptic and postsynaptic recordings from the mouse calyx of Held, we examined the effect of acute selective pharmacological inhibition of glycolysis or mitochondrial OxPhos on multiple mechanisms regulating presynaptic function. Inhibition of glycolysis via glucose depletion and iodoacetic acid (1 mM) treatment, but not mitochondrial OxPhos, rapidly altered transmission, resulting in highly variable, oscillating responses. At reduced temperature, this same treatment attenuated synaptic transmission because of a smaller and broader presynaptic action potential (AP) waveform. We show via experimental manipulation and ion channel modeling that the altered AP waveform results in smaller Ca influx, resulting in attenuated excitatory postsynaptic currents (EPSCs). In contrast, inhibition of mitochondria-derived ATP production via extracellular pyruvate depletion and bath-applied oligomycin (1 μM) had no significant effect on Ca influx and did not alter the AP waveform within the same time frame (up to 30 min), and the resultant EPSC remained unaffected. Glycolysis, but not mitochondrial OxPhos, is thus required to maintain basal synaptic transmission at the presynaptic terminal. We propose that glycolytic enzymes are closely apposed to ATP-dependent ion pumps on the presynaptic membrane. Our results indicate a novel mechanism for the effect of hypoglycemia on neurotransmission. Attenuated transmission likely results from a single presynaptic mechanism at reduced temperature: a slower, smaller AP, before and independent of any effect on synaptic vesicle release or receptor activity.

Ionotropic activation of NMDA receptors (NMDARs) requires agonist glutamate and co-agonist glycine. Here we show that glycine enhances the activation of cell survival-promoting kinase Akt in cultured cortical neurons in which both the channel activity of NMDARs and the glycine receptors are pre-inhibited. The effect of glycine is reduced by shRNA-mediated knockdown of GluN2A subunit-containing NMDARs (GluN2ARs), suggesting that a non-ionotropic activity of GluN2ARs mediates glycine-induced Akt activation. In support of this finding, glycine enhances Akt activation in HEK293 cells over-expressing GluN2ARs. The effect of glycine on Akt activation is sensitive to the antagonist of glycine-GluN1 binding site. As a functional consequence, glycine protects against excitotoxicity-induced neuronal death through the non-ionotropic activity of GluN2ARs and the neuroprotective effect is attenuated by Akt inhibition. Thus, this study reveals an unexpected role of glycine in eliciting a non-ionotropic activity of GluN2ARs to confer neuroprotection via Akt activation.

GluA2-lacking Ca 2ϩ -permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca 2ϩ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca 2ϩ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ϳ80% of the AMPAR population are permeable to Ca 2ϩ at immature P4 -P5 synapses. However, after the onset of hearing, Ca 2ϩ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18 -P22 and P30 -P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30 -P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca 2ϩ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca 2ϩ permeable. However, we find that Ca 2ϩ -permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca 2ϩ -permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.

Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs’ ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.